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Mutations in the amyloid-β protein precursor gene (AβPP), the presenilin 1 gene (PSEN1) or the presenilin 2 gene (PSEN2) that increase production of the AβPP-derived peptide Aβ42 cause early-onset Alzheimer's disease. Rodent models of the disease show that further increase in Aβ42 production and earlier brain pathology can be obtained by coexpressing AβPP and PSEN1 mutations. To generate such elevated Aβ42 level in a large animal model, we produced Göttingen minipigs carrying in their genome one copy of a human PSEN1 cDNA with the Met146Ile (PSEN1M146I) mutation and three copies of a human AβPP695 cDNA with the Lys670Asn/Met671Leu (AβPPsw) double-mutation. Both transgenes were expressed in fibroblasts and in the brain, and their respective proteins were processed normally. Immunohistochemical staining with Aβ42-specific antibodies detected intraneuronal accumulation of Aβ42 in brains from a 10- and an 18-month-old pig. Such accumulation may represent an early event in the pathogenesis of Alzheimer's disease.

Autosomal dominant familial Alzheimer's disease (AD) is associated with mutations in the AβPP, PSEN1, and PSEN2 genes. The clinical phenotype associated with AβPP mutations is mainly characterized by dementia or by strokes related to cerebral amyloid angiopathy (CAA). We present a comprehensive clinical, neuropathological, genetic, and biochemical study on a patient affected by familial AD associated with the I716F mutation in the AβPP gene. The clinical phenotype was characterized by early age of onset of 47 years, and rapidly progressive cerebellar ataxia, myoclonic jerks, rigidity, and dementia reminiscent of Creutzfeldt-Jakob disease (CJD), followed by a prolonged persistent vegetative state. Neuropathological evaluation of the proband revealed AD-related pathology but also α-synucleinopathy compatible with dementia with Lewy bodies neocortical stage or Parkinson's disease corresponding to Braak stage 6. Tau-pathology in the form of neurofibrillary degeneration corresponded to stage VI according to the Braak classification. The severe Aβ pathology included CAA, numerous plaques, and deposition of N-truncated pyroglutamate-modified Aβ peptides. Remarkably, pyroglutamate Aβ oligomers were also present intracellularly in Purkinje cells corresponding to the ataxic phenotype. The detection of a CJD-like phenotype expands the spectrum of clinical presentations associated with familial AD. Our study supports the concept that the neuropathology of familial AD expands beyond the classical AD-related pathology as defined by plaques and tangles. Finally, we provide evidence for the first time that oligomeric pyroglutamate Aβ is present in a specific pattern correlating with the clinical symptoms of a patient with AβPP I716F mutation.

Background: The Arctic mutation (p.E693G/p.E22G)fs within the β-amyloid (Aβ) region of the β-amyloid precursor protein gene causes an autosomal dominant disease with clinical picture of typical Alzheimer's disease. Here we report the special character of Arctic AD neuropathology in four deceased patients., Results: Aβ deposition in the brains was wide-spread (Thal phase 5) and profuse. Virtually all parenchymal deposits were composed of non-fibrillar, Congo red negative Aβ aggregates. Congo red only stained angiopathic vessels. Mass spectrometric analyses showed that Aβ deposits contained variably truncated and modified wild type and mutated Aβ species. In three of four Arctic AD brains, most cerebral cortical plaques appeared targetoid with centres containing C-terminally (beyond aa 40) and variably N-terminally truncated Aβ surrounded by coronas immunopositive for Aβx-42. In the fourth patient plaque centres contained almost no Aβ making the plaques ring-shaped. The architectural pattern of plaques also varied between different anatomic regions. Tau pathology corresponded to Braak stage VI, and appeared mainly as delicate neuropil threads (NT) enriched within Aβ plaques. Dystrophic neurites were scarce, while neurofibrillary tangles were relatively common. Neuronal perikarya within the Aβ plaques appeared relatively intact., Conclusions: In Arctic AD brain differentially truncated abundant Aβ is deposited in plaques of variable numbers and shapes in different regions of the brain (including exceptional targetoid plaques in neocortex). The extracellular non-fibrillar Aβ does not seem to cause overt damage to adjacent neurons or to induce formation of neurofibrillary tangles, supporting the view that intracellular Aβ oligomers are more neurotoxic than extracellular Aβ deposits. However, the enrichment of NTs within plaques suggests some degree of intra-plaque axonal damage including accumulation of hp-tau, which may impair axoplasmic transport, and thereby contribute to synaptic loss. Finally, similarly as the cotton wool plaques in AD resulting from exon 9 deletion in the presenilin-1 gene, the Arctic plaques induced only modest glial and inflammatory tissue reaction.

Increasing evidence suggests an important function of the β-amyloid precursor protein (APP) in malignant disease in humans; however, the biological basis for this evidence is not well understood at present. To understand the role of APP in transformed pluripotent stem cells, we studied its expression levels in human testicular germ cell tumors using patient tissues, model cell lines, and an established xenograft mouse model. In the present study, we demonstrate the cooperative expression of APP with prominent pluripotency-related genes such as Sox2, NANOG, and POU5F1 (Oct3/4). The closest homologue family member, APLP2, showed no correlation to these stem cell factors. In addition, treatment with histone deacetylase (HDAC) inhibitors suppressed the levels of APP and stem cell markers. Loss of pluripotency, either spontaneously or as a consequence of treatment with an HDAC inhibitor, was accompanied by decreased APP protein levels both in vitro and in vivo. These observations suggest that APP represents a novel and specific biomarker in human transformed pluripotent stem cells that can be selectively modulated by HDAC inhibitors., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The triple-transgenic Alzheimer's disease (AD) mouse model, 3xTg-AD, played an important role in supporting the intraneuronal amyloid-β (Aβ) hypothesis in AD. However, recent evidence claims that the 3xTg-AD mice accumulate amyloid-β protein precursor (AβPP) instead of Aβ within neurons. In the present report, we re-investigated the 3xTg-AD mouse model and confirmed recent findings of age-dependent AβPP accumulations. In addition, intraneuronal Aβ was detected mainly in the neocortex using conformation-specific as well as antibodies directed against Aβ neo-epitopes. In contrast to previous work, however, only minor levels of intraneuronal Aβ were detected in the CA1 region of the hippocampus in aged 3xTg-AD mice.

Gangliosides are important players for controlling neuronal function and are directly involved in AD pathology. They are among the most potent stimulators of Aβ production, are enriched in amyloid plaques and bind amyloid beta (Aβ). However, the molecular mechanisms linking gangliosides with AD are unknown. Here we identified the previously unknown function of the amyloid precursor protein (APP), specifically its cleavage products Aβ and the APP intracellular domain (AICD), of regulating GD3-synthase (GD3S). Since GD3S is the key enzyme converting a- to b-series gangliosides, it therefore plays a major role in controlling the levels of major brain gangliosides. This regulation occurs by two separate and additive mechanisms. The first mechanism directly targets the enzymatic activity of GD3S: Upon binding of Aβ to the ganglioside GM3, the immediate substrate of the GD3S, enzymatic turnover of GM3 by GD3S was strongly reduced. The second mechanism targets GD3S expression. APP cleavage results, in addition to Aβ release, in the release of AICD, a known candidate for gene transcriptional regulation. AICD strongly down regulated GD3S transcription and knock-in of an AICD deletion mutant of APP in vivo, or knock-down of Fe65 in neuroblastoma cells, was sufficient to abrogate normal GD3S functionality. Equally, knock-out of the presenilin genes, presenilin 1 and presenilin 2, essential for Aβ and AICD production, or of APP itself, increased GD3S activity and expression and consequently resulted in a major shift of a- to b-series gangliosides. In addition to GD3S regulation by APP processing, gangliosides in turn altered APP cleavage. GM3 decreased, whereas the ganglioside GD3, the GD3S product, increased Aβ production, resulting in a regulatory feedback cycle, directly linking ganglioside metabolism with APP processing and Aβ generation. A central aspect of this homeostatic control is the reduction of GD3S activity via an Aβ-GM3 complex and AICD-mediated repression of GD3S transcription.

Loss of cholinergic neurons in the Nucleus Basalis of Meynert in Alzheimer's disease (AD) patients was one of the first discoveries of neuron loss in AD. Despite an intense focus on the cholinergic system in AD, the reason for this cholinergic neuron loss is yet unknown. In the present study we examined Abeta-induced pathology and neuron loss in the cholinergic system of the bigenic APP/PS1KI mouse model. Expression of the APP transgene was found in ChAT-positive neurons of motor nuclei accompanied by robust intracellular Abeta accumulation, whereas no APP expressing neurons and thus no intracellular Abeta accumulation were found in neither the forebrain or pons complexes, nor in the caudate putamen. This expression pattern was used as a model system to study the effect of intra- and extracellular Abeta accumulation on neuron loss in the cholinergic system. Stereological quantification revealed a loss of ChAT-positive neurons in APP/PS1KI mice only in the motor nuclei Mo5 and 7N accumulating intracellular Abeta. This study supports the hypothesis of intracellular Abeta accumulation as an early pathological alteration contributing to cell death in AD., (Copyright 2008 Elsevier Inc. All rights reserved.)

The beta-amyloid precursor protein (APP) represents a type I transmembrane glycoprotein that is ubiquitously expressed. In the brain, it is a key player in the molecular pathogenesis of Alzheimer disease. Its physiological function is however less well understood. Previous studies showed that APP is up-regulated in prostate, colon, pancreatic tumor, and oral squamous cell carcinoma. In this study, we show that APP has an essential role in growth control of pancreatic and colon cancer. Abundant APP staining was found in human pancreatic adenocarcinoma and colon cancer tissue. Interestingly, treating pancreatic and colon cancer cells with valproic acid (VPA, 2-propylpentanoic acid), a known histone deacetylase (HDAC) inhibitor, leads to up-regulation of GRP78, an endoplasmic reticulum chaperone immunoglobulin-binding protein. GRP78 is involved in APP maturation and inhibition of tumor cell growth by down-regulation of APP and secreted soluble APPalpha. Trichostatin A, a pan-HDAC inhibitor, also lowered APP and increased GRP78 levels. In contrast, treating cells with valpromide, a VPA derivative lacking HDAC inhibitory properties, had no effect on APP levels. VPA did not modify the level of epidermal growth factor receptor, another type I transmembrane protein, and APLP2, a member of the APP family, demonstrating the specificity of the VPA effect on APP. Small interfering RNA-mediated knockdown of APP also resulted in significantly decreased cell growth. Based on these observations, the data suggest that APP down-regulation via HDAC inhibition provides a novel mechanism for pancreatic and colon cancer therapy.

Abeta accumulation has an important function in the etiology of Alzheimer's disease (AD) with its typical clinical symptoms, like memory impairment and changes in personality. However, the mode of this toxic activity is still a matter of scientific debate. We used the APP/PS1KI mouse model for AD, because it is the only model so far which develops 50% hippocampal CA1 neuron loss at the age of 1 year. Previously, we have shown that this model develops severe learning deficits occurring much earlier at the age of 6 months. This observation prompted us to study the anatomical and cellular basis at this time point in more detail. In the current report, we observed that at 6 months of age there is already a 33% CA1 neuron loss and an 18% atrophy of the hippocampus, together with a drastic reduction of long-term potentiation and disrupted paired pulse facilitation. Interestingly, at 4 months of age, there was no long-term potentiation deficit in CA1. This was accompanied by reduced levels of pre- and post-synaptic markers. We also observed that intraneuronal and total amount of different Abeta peptides including N-modified, fibrillar and oligomeric Abeta species increased and coincided well with CA1 neuron loss. Overall, these data provide the basis for the observed robust working memory deficits in this mouse model for AD at 6 months of age.

The accumulation of beta-amyloid (A beta) plaques and neurofibrillary tangles consisting of hyperphosphorylated tau protein are pathological features of Alzheimer's disease (AD) commonly modeled in mice using known human familial mutations; however, the loss of neurons also found to occur in AD is rarely observed in such models. The mechanism of neuron degeneration remains unclear but is of great interest as it is very likely an important factor for the onset of adverse memory deficits occurring in individuals with AD. The role of A beta in the neuronal degeneration is a matter of controversial debates. In the present study we investigated the impact of extracellular plaque A beta versus intraneuronal A beta on neuronal cell death. The thalamus and the frontal cortex of the APP/PS1KI mouse model were chosen for stereological quantification representing regions with plaques only (thalamus) or plaques as well as intraneuronal A beta (frontal cortex). A loss of neurons was found in the frontal cortex at the age of 6 months coinciding with the decrease of intraneuronal immunoreactivity, suggesting that the neurons with early intraneuronal A beta accumulation were lost. Strikingly, no neuron loss was observed in the thalamus despite the development of abundant plaque pathology with levels comparable to the frontal cortex. This study suggests that plaques have no effect on neuron death whereas accumulation of intraneuronal A beta may be an early transient pathological event leading to neuron loss in AD.

Loss of neurons in the hippocampus and other brain regions is, besides the occurrence of plaques and tangles, a neuropathological feature of Alzheimer's disease (AD). In recent years a plethora of transgenic mouse models overexpressing mutant amyloid precursor protein (APP) has been developed, which represent valuable research tools. Whereas extracellular plaque pathology is a common feature of these models, neuronal loss is a rather rare characteristic. In the present study, we quantified the number of neurons in the dentate gyrus granule layer (GCL) in 2- and 12-month-old APP/PS1KI mice, a mouse model that has been previously shown to have significant loss of neurons in the CA1 layer of the hippocampus. Stereological analysis revealed a strongly significant decrease of GCLs in aged APP/PS1KI mice, compared to age-matched PS1KI control animals (-44%), however, the volume of the GCL was not different.

The APP/PS1ki mouse model for Alzheimer's disease (AD) exhibits robust brain and spinal cord axonal degeneration and hippocampal CA1 neuron loss starting at 6 months of age. It expresses human mutant APP751 with the Swedish and London mutations together with two FAD-linked knocked-in mutations (PS1 M233T and PS1 L235P) in the murine PS1 gene. The present report covers a phenotypical analysis of this model using either behavioral tests for working memory and motor performance, as well as an analysis of weight development and body shape. At the age of 6 months, a dramatic, age-dependent change in all of these properties and characteristics was observed, accompanied by a significantly reduced ability to perform working memory and motor tasks. The APP/PS1ki mice were smaller and showed development of a thoracolumbar kyphosis, together with an incremental loss of body weight. While 2-month-old APP/PS1ki mice were inconspicuous in all of these tasks and properties, there is a massive age-related impairment in all tested behavioral paradigms. We have previously reported robust axonal degeneration in brain and spinal cord, as well as abundant hippocampal CA1 neuron loss starting at 6 months of age in the APP/PS1ki mouse model, which coincides with the onset of motor and memory deficits described in the present report.

Sortilin-related receptor with A-type repeats (SORLA) is a sorting receptor that impairs processing of amyloid precursor protein (APP) to soluble (s) APP and to the amyloid beta-peptide in cultured neurons and is poorly expressed in patients with Alzheimer disease (AD). Here, we evaluated the consequences of Sorla gene defects on brain anatomy and function using mouse models of receptor deficiency. In line with a protective role for SORLA in APP metabolism, lack of the receptor results in increased amyloidogenic processing of endogenous APP and in aggravated plaque deposition when introduced into PDAPP mice expressing mutant human APP. Surprisingly, increased levels of sAPP caused by receptor deficiency correlate with pro-found stimulation of neuronal ERK signaling and with enhanced neurogenesis, providing in vivo support for neurotrophic functions of sAPP. Our data document a role for SORLA not only in control of plaque burden but also in APP-dependent neuronal signaling and suggest a molecular explanation for increased neurogenesis observed in some AD patients.

Accumulating evidence points to an important role of intraneuronal beta-amyloid (Abeta) in the development of Alzheimer's disease (AD), with its typical clinical symptoms like memory impairment and changes in personality. We have previously reported on the Abeta precursor protein and presenilin-1 knock-out (APP/PS1KI) mouse model with abundant intraneuronal Abeta(42) accumulation and a 50% loss of CA1 neurons at 10 months of age. In addition, we observed reduced short- and long-term synaptic plasticity, hippocampal neuron loss, and reduced performance in a working memory task. These observations support a pivotal role of intraneuronal Abeta accumulation as a principal pathological trigger in AD., (2008 S. Karger AG, Basel)

Cholesterol has been implicated to play an important role in the generation of Abeta peptides, which are the main component of beta-amyloid plaques in the brains of patients suffering from Alzheimer's disease (AD). Epidemiological data implicate that lowering cholesterol levels has beneficial effects on the extent of beta-amyloid pathology. Thus therapeutic intervention using cholesterol lowering drugs like statins seems to be a promising approach. A couple of studies, in vitro or in vivo by the use of AD transgenic mouse models, focused on the manipulation of cholesterol levels and the resulting effects on Abeta generation. In contrast, there is not much known about the effect of the amyloid precursor protein (APP) on cholesterol levels. In the present report, we transfected human neuroblastoma cells with human APP695 and compared cellular cholesterol levels with the respective levels in Mock-transfected control cells. Furthermore, we determined the levels of diverse cholesterol precursors and metabolites using gas chromatography-mass spectrometry (GC-MS). Significant differences in the levels of the respective cholesterol precursors were observed, whereas inhibition of gamma-secretase activity by the gamma-secretase inhibitor DAPT did not have a significant effect on cellular cholesterol metabolism.

There is mounting evidence that the amyloid precursor protein (APP), the key protein in Alzheimer's disease (AD) is involved in the copper (Cu) homeostasis in the brain. Conflicting results about the potential use of dietary Cu and clioquinol (CQ), a known Cu chelator, have been reported using APP transgenic mice. Previously, in vitro studies have demonstrated that CQ can act as a Cu transporter. To analyze the potential function of CQ as a Cu transporter in vivo, the nutritional effect of Cu and CQ was analyzed in young APP transgenic mice and nontransgenics with food pellets containing either Cu, CQ, Cu plus CQ (Cu + CQ), or without addition of supplements (control). The offspring were fed with corresponding food pellets until the age of 14 weeks. We observed an increased lethality of APP transgenics upon CQ treatment, which could be rescued by a co-treatment with Cu. The exposure of Cu + CQ led to a modest but significant increase in cerebral Cu levels, most likely due to an enhanced transport of CQ-Cu complexes. In CQ or Cu + CQ treatment groups, the plasma levels of Cu, zinc, and iron were reduced in all animals; moreover, Cu treatment alone reduced only plasma iron levels. We conclude not only that CQ has certain toxicity but also that the chelating effect, perhaps, plays a secondary role with respect to its properties as an intracellular Cu transporter, thus, counteracting the supposed therapeutic effects of CQ as an agent for chelating therapy in AD.

While axonopathy is a prominent feature in a variety of neurodegenerative diseases, it has been largely neglected in Alzheimer's disease (AD), despite the observation of frequent motoric deficits in AD patients. In the present report we used transgenic mice overexpressing human mutant beta-amyloid precursor protein (APP(751SL)) and presenilin-1 (PS1(M146L)) that exhibit elevated intraneuronal Abeta42 levels. We observed abundant age-dependent axonopathy in the spinal cord: axons immunopositive for ubiquitin in the dorsal column; axonal swellings (spheroids) which accumulated APP, neurofilament, and ubiquitin; as well as myelin ovoid structures, which serve as markers for nerve fiber degeneration in both white and gray matter. Both descending and ascending axonal tracts in white matter were affected. Neuritic plaques also developed in an age-dependent manner starting in the cervical region. Furthermore, early intraneuronal Abeta was detected in some but not all motor neurons before plaque formation. In the present APP/PS1 transgenic mouse model we could show for the first time that elevated intracellular Abeta levels lead to an axonopathy characterized by the formation of axonal spheroids and myelin ovoids. The same pathological alterations are known from AD patients or transgenic models overexpressing Tau or ApoE, however, these disturbances in axonal transport occur in the absence of any signs of concomitant Tau pathology. This strengthens the prevailing amyloid hypothesis as a primary trigger of AD-typical pathological alterations.

AbetaPP is involved in Cu homeostasis in mouse and man. In vitro observations and in vivo data obtained from AbetaPP mouse models at least provide strong evidence that AbetaPP and Abeta overproduction enables intracellular Cu to be transported out of the cell. The increased Cu efflux seems to lead to a Cu deficiency and a subsequently reduced SOD-1 activity. Studies have shown that a disturbed metal-ion homeostasis with elevated serum Cu levels occurs in Alzheimer and Down's patients and lowered levels in post-mortem AD brain. We conclude that bioavailable Cu has beneficial and specific effects in Alzheimer's disease transgenic mice, and suggest that our observation can be regarded as a proof-of-concept for a prophylactic approach to overcome the observed CNS Cu deficiency in the brain of Alzheimer's disease patients.

Neuron and synapse loss are important features of the neuropathology of Alzheimer's disease (AD). Recently, we observed substantial age-related hippocampal neuron loss in APP751SL/PS1M146L transgenic mice but not in PS1M146L mice. Here, we investigated APP751SL mice, PS1M146L mice, and APP751SL/PS1M146L mice for age-related alterations in synaptic integrity within hippocampal stratum moleculare of the dentate gyrus (SM), stratum lucidum of area CA3 (SL), and stratum radiatum of area CA1-2 (SR) by analyzing densities and numbers of synaptophysin-immunoreactive presynaptic boutons (SIPBs). Wild-type mice, APP751SL mice and PS1M146L mice showed similar amounts of age-related SIPB loss within SM, and no SIPB loss within SL. Both APP751SL mice and PS1M146L mice showed age-related SIPB loss within SR. Importantly, APP751SL/PS1M146L) mice displayed the severest age-related SIPB loss within SM, SL, and SR, even in regions free of extracellular Abeta deposits. Together, these mouse models offer a unique framework to study the impact of several molecular and cellular events caused by mutant APP and/or mutant PS1 on age-related alterations in synaptic integrity. The observation of age-related SIPB loss within SR of PS1M146L mice supports a role of mutant PS1 in neurodegeneration apart from its contribution to alterations in Abeta generation.

Oxidative stress plays an important role in the pathogenesis of Alzheimer's disease. To determine which mechanisms cause the origin of oxidative damage, we analyzed enzymatic antioxidant defense (Cu/Zn-superoxide dismutase Cu/Zn-SOD, glutathione peroxidase GPx and glutathione reductase GR) and lipid peroxidation products malondialdehyde MDA and 4-hydroxynonenal HNE in two different APP transgenic mouse models at 3-4 and 12-15 months of age. No changes in any parameter were observed in brains from PDGF-APP695(SDL) mice, which have low levels of Abeta and no plaque load. In contrast, Thy1-APP751(SL) mice show high Abeta accumulation with aging and plaques from an age of 6 months. In brains of these mice, HNE levels were increased at 3 months (female transgenic mice) and at 12 months (both gender), that is, before and after plaque deposition, and the activity of Cu/Zn-SOD was reduced. Interestingly, beta-amyloidogenic cleavage of APP was increased in female Thy1-APP751(SL) mice, which also showed increased HNE levels with simultaneously reduced Cu/Zn-SOD activity earlier than male Thy1-APP751(SL) mice. Our results demonstrate that impaired Cu/Zn-SOD activity contributes to oxidative damage in Thy1-APP751(SL) transgenic mice, and these findings are closely linked to increased beta-amyloidogenic cleavage of APP.

The key protein in Alzheimer's disease, the amyloid precursor protein (APP), is a ubiquitously expressed copper-binding glycoprotein that gives rise to the Abeta amyloid peptide. Whereas overexpression of APP results in significantly reduced brain copper levels in three different lines of transgenic mice, knock-out animals revealed increased copper levels. A provoked rise in peripheral levels of copper reduced concentrations of soluble amyloid peptides and resulted in fewer pathogenic Abeta plaques. Contradictory evidence has been provided by the efficacy of copper chelation treatment with the drug clioquinol. Using a yeast model system, we show that adding clioquinol to the yeast culture medium drastically increased the intracellular copper concentration but there was no significant effect observed on zinc levels. This finding suggests that clioquinol can act therapeutically by changing the distribution of copper or facilitating copper uptake rather than by decreasing copper levels. The overexpression of the human APP or APLP2 extracellular domains but not the extracellular domain of APLP1 decreased intracellular copper levels. The expression of a mutant APP deficient for copper binding increased intracellular copper levels several-fold. These data uncover a novel biological function for APP and APLP2 in copper efflux and provide a new conceptual framework for the formerly diverging theories of copper supplementation and chelation in the treatment of Alzheimer's disease.

Beta-site APP-cleaving enzyme (BACE) is a membrane-bound aspartyl protease with no strict primary preference for cleavage. The molecular mechanisms that link the gamma-secretase multicomponent amyloid precursor protein (APP) processing complex to biochemical properties of BACE generating the N terminus of the amyloid beta-peptide have not, as yet, been identified. We found that in human brain tissue, BACE occurred as a dimer. The overall stability of the BACE homodimer was based on intermolecular interactions that were not affected by high salt, nonionic detergents or reducing conditions. BACE homodimers could only partially be separated even under strong denaturing conditions and revealed dramatic differences in the surface charge distribution compared with the monomer. In contrast, the soluble ectodomain of truncated BACE revealed a seemingly lower avidity to the prototypic aspartate protease inhibitor pepstatin and exclusively occurred in the monomeric form. Immunocytochemical studies colocalized APP and BACE in the plasma membrane of cells expressing endogenous levels of BACE and overexpressing APP. In cells that were cotransfected with APP and a putative active site D289A mutant of BACE, colocalization persisted. Remaining enzyme activity was found to be attributable to the mutant protease. Accordingly, inactivation of the carboxyl-terminal active site motif of BACE without an impairment of overall enzyme activity suggests that the enzyme may act as a dimer. Thus, homodimerization of BACE may help the enzyme to acquire specific mechanisms to associate with its substrates to exert catalytic activity.

The Cu-binding beta-amyloid precursor protein (APP), and the amyloid Abeta peptide have been proposed to play a role in physiological metal regulation. There is accumulating evidence of an unbalanced Cu homeostasis with a causative or diagnostic link to Alzheimer's disease. Whereas elevated Cu levels are observed in APP knockout mice, APP overexpression results in reduced Cu in transgenic mouse brain. Moreover, Cu induces a decrease in Abeta levels in APP-transfected cells in vitro. To investigate the influence of bioavailable Cu, transgenic APP23 mice received an oral treatment with Cu-supplemented sucrose-sweetened drinking water (1). Chronic APP overexpression per se reduced superoxide dismutase 1 activity in transgenic mouse brain, which could be restored to normal levels after Cu treatment (2). A significant increase of brain Cu indicated its bioavailability on Cu treatment in APP23 mice, whereas Cu levels remained unaffected in littermate controls (3). Cu treatment lowered endogenous CNS Abeta before a detectable reduction of amyloid plaques. Thus, APP23 mice reveal APP-induced alterations linked to Cu homeostasis, which can be reversed by addition of dietary Cu.

Several novel transgenic mouse models expressing different mutant APPs in combination with mutant PS1 have been developed. These models have been analyzed to investigate the formation and progressive alterations of dystrophic neurites (DNs) in relation to Abeta deposits. In the most aggressive model, Abeta deposits appear as early as 2.5 months of age. Maturation of DNs was qualitatively quite similar among models and in some respect reminiscent of human AD pathology. From the onset of deposition, most if not all Abeta deposits were decorated with a high number of APP-, ubiquitin-, and MnSOD-immunoreactive DNs. Phosphorylated Tau DNs, however, appeared at a much slower rate and were more restricted. Mitochondrial dysfunction markers were observed in DNs: the frequency and the density per deposit of DNs accumulating cytochrome c, cytochrome oxidase 1, and Bax progressively increased with age. Later, the burden of reactive DNs was reduced around large compact/mature deposits. In addition, the previously described phenomenon of early intraneuronal Abeta accumulation in our models was associated with altered expression of APP protein as well as oxidative and mitochondrial stress markers occasionally in individual neurons. The present study demonstrates that oxidative and mitochondrial stress factors are present at several phases of Abeta pathology progression, confirming the neuronal dysfunction in APP transgenic mice.

Previous studies in the literature have resulted in conflicting reports on the potential neurotoxicity of the beta-cleaved Alzheimer's disease C-terminal fragment (beta-CTF) of beta-amyloid precursor protein in vivo. To readdress this question by rigorous quantitative methods, we analyzed transgenic mice expressing human beta-CTF with the I45F mutation (SPA4CT) under control of the prion protein promoter by stereological techniques. The transgene was expressed in hippocampus and cortex in large pyramidal neurons and in dentate gyrus granule cells. Proteolytic processing of beta-CTF released Abeta. However, most of it remained uncleaved. Neurodegeneration was evaluated by investigating the numbers of hippocampal pyramidal and granule neurons, as well as the number of synaptophysin-immunopositive presynaptic boutons in the hippocampus of 15-month-old SPA4CT mice with design-based stereological techniques. The analyses showed that a fourfold higher expression of the transgene compared to murine APP levels had no effect on the numbers of both neurons and synaptophysin-immunopositive presynaptic boutons. These data implicate that expression of beta-CTF per se is not neurotoxic, and that other mechanisms are responsible for the neurotoxic events in Alzheimer's disease brain., (Copyright 2003 Elsevier Science (USA))

The amyloid precursor protein (APP) copper-binding domain (CuBD) has been shown to reduce Cu(II) to Cu(I) and to mediate copper-induced oxidation in vitro. However, little is known about copper binding to the homologous domains of APP and APP family paralogs and orthologs (including amyloid precursor-like proteins from Drosophila melanogaster, Xenopus laevis, and Caenorhabditis elegans) and their effects on Cu-induced oxidation and Cu(I) formation. Here, we show that APP homologues with and without conserved histidine residues at positions 147, 149, and 151 all bind Cu(II). Oxidized peptides were the kinetically favored products of the redox reaction of CuBDs promoting the reduction of Cu(II) to Cu(I). These results reveal a molecular phylogeny-based divergence that has taken place between the ancestral Drosophila APPL and C. elegans APL-1 and the recently evolved APP lineage of CuBDs. Whereas higher species CuBDs have a decreased affinity for Cu(II) and high Cu(II) reducing activities, ancestral CuBDs form very tight binding sites for Cu(II) ions and have low Cu(II) reducing activities. Thus, the APP lineage displays a gain in activity toward promoting Cu(II) reduction and Cu(I) release. The findings suggests that the Cu(II)-binding equilibrium at the phylogenetic stage of Drosophila APPL and C. elegans APL-1 is shifted from the exchangeable Cu(II) pool to the tightly bound, nonexchangeable pool and that ancestral CuBDs may exert antioxidation activities in vivo. The more recently evolved homologues of human APP appear to take advantage of unique redox properties for yet unknown biological functions.

Neuropil deposition of beta-amyloid peptides A beta40 and A beta42 is believed to be the key event in the neurodegenerative processes of Alzheimer's disease (AD). Since A beta seems to carry a transport signal that is required for axonal sorting of its precursor beta-amyloid precursor protein (APP), we studied the intraneuronal staining profile of A beta peptides in a transgenic mouse model expressing human mutant APP751 (KM670/671NL and V7171) and human mutant presenilin-1 (PS-1 M146L) in neurons. Using surface plasmon resonance we analyzed the A beta antibodies and defined their binding profile to APP, A beta40 and A beta42. Immunohistochemical staining revealed that intraneuronal A beta40 and A beta42 staining preceded plaque deposition, which started at 3 months of age. A beta was observed in the somatodendritic and axonal compartments of many neurons. Interestingly, the striatum, which lacks transgenic APP expression harbored many plaques at 10 months of age. This is most likely due to an APP/A beta transport problem and may be a model region to study APP/A beta trafficking as an early pathological event.

Cholesterol is implicated to play a role in Alzheimer disease pathology. Therefore, the concentrations of cholesterol, its precursors, and its degradation products in brain homogenates of aging wild-type and beta-amyloid precursor protein transgenic mice carrying the Swedish mutation (APP23) were analyzed. Among the sterols measured, lanosterol is the first common intermediate of two different pathways, which use either desmosterol or lathosterol as the predominant precursors for de novo synthesis of brain cholesterol. In young mice, cholesterol is mainly synthesized via the desmosterol pathway, while in aged mice, lathosterol is the major precursor. 24S-hydroxycholesterol (cerebrosterol), which plays a key role in the removal of cholesterol from the brain, modestly increased during aging. No differences in the levels of cholesterol, its precursors, or its metabolites were found between wild-type and APP23 transgenic mice. Moreover, the levels of the exogenous plant sterols campesterol and sitosterol were significantly elevated in the brains of APP23 animals at age 12 and 18 months. This time point coincides with abundant plaque formation.

Oxidative stress was presented to play an important role in the pathogenesis of Alzheimer's disease (AD), especially in the early evolution of AD amyloidogenesis and not only as a consequence thereof. The effect of oxidative stress catalysed by transition metals appears to have a critical relevance in AD. Metal-ion homeostasis is severely dysregulated in AD and it was found that experimentally induced disturbances in the homeostasis of Zn(II) and Cu(II) affect the amyloid precursor protein (APP) metabolism. APP itself binds Zn(II) and Cu(II) at nanomolar concentrations and an altered APP metabolism or expression level is believed to result in neurotoxic processes.

The pathogenesis of Alzheimer's disease (AD) is believed to be closely dependent on deposits of neurotoxic amyloid-β peptides (Aβ), which become abundantly present throughout the central nervous system in advanced stages of the disease. The different Aβ peptides existing are generated by subsequent cleavage of the amyloid-β protein precursor (AβPP) and may vary in length and differ at their C-terminus. Despite extensive studies on the most prevalent species Aβ40 and Aβ42, Aβ peptides with other C-termini such as Aβ38 have not received much attention. In the present study, we used a highly specific and sensitive antibody against Aβ38 to analyze the distribution of this Aβ species in cases of sporadic and familial AD, as well as in the brains of a series of established transgenic AD mouse models. We found Aβ38 to be present as vascular deposits in the brains of the majority of sporadic AD cases, whereas it is largely absent in non-demented control cases. Aβ38-positive extracellular plaques were virtually limited to familial cases. Interestingly we observed Aβ38-positive plaques not only among familial cases due to AβPP mutations, but also in cases of familial AD caused by presenilin (PSEN) mutations. Furthermore we demonstrate that Aβ38 deposits in the form of extracellular plaques are common in several AD transgenic mouse models carrying either only AβPP, or combinations of AβPP, PSEN1, and tau transgenes. [ABSTRACT FROM AUTHOR]

Background According to the modified amyloid hypothesis the main event in the pathogenesis of Alzheimer's disease (AD) is the deposition of neurotoxic amyloid β-peptide (Aβ) within neurons. Additionally to full-length peptides, a great diversity of N-truncated Aβ variants is derived from the larger amyloid precursor protein (APP). Vast evidence suggests that Aβx-42 isoforms play an important role triggering neurodegeneration due to its high abundance, amyloidogenic propensity and toxicity. Although N-truncated and Aβx-42 species have been pointed as crucial players in AD etiology, the Aβ5-x isoforms have not received much attention. Results The present study is the first to show immunohistochemical evidence of Aβ5-x in familial cases of AD (FAD) and its distribution in APP/PS1KI, 5XFAD and 3xTG transgenic mouse models. In order to probe Aβ5-x peptides we generated the AB5-3 antibody. Positive plaques and congophilic amyloid angiopathy (CAA) were observed among all the FAD cases tested carrying either APP or presenilin 1 (PS1) mutations and most of the sporadic cases of AD (SAD). Different patterns of Aβ5-x distribution were found in the mouse models carrying different combinations of autosomal mutations in the APP, PS1 and Tau genes. All of them showed extracellular Aβ deposits but none CAA. Additionally, they were all affected by a severe amyloid pathology in the hippocampus among other areas. Interestingly, neither 5XFAD nor APP/PS1KI showed any evidence for intraneuronal Aβ5-x. Conclusions Different degrees of Aβ5-x accumulations can be found in the transgenic AD mouse models and human cases expressing the sporadic or the familial form of the disease. Due to the lack of intracellular Aβ5-x, these isoforms might not be contributing to early mechanisms in the cascade of events triggering AD pathology. Brain sections obtained from SAD cases showed higher Aβ5-x-immunoreactivity in vascular deposits than in extracellular plaques, while both are equally important in the FAD cases. The difference may rely on alternative mechanisms involving Aβ5-x peptides and operating in a divergent way in the late and early onset forms of the disease. [ABSTRACT FROM AUTHOR]